During the last decade, regulatory T (Treg) cells have emerged as key regulators of immune responses to self-antigens, allergens, commensal microbiota, infectious agents and tumors. Our previous work and work by others showed that transcriptional factor FoxpS serves as a lineage specification factor of Treg cells (Fontenot et al., 2003; 2005; Hori et al., 2003; Khattri et al., 2003). We also demonstrated that paucity of Treg cells due to loss-of-function mutations of the Foxp3 gene is responsible for highly aggressive, fatal systemic immune-mediated inflammatory lesions in mice and men (Fontenot et al., 2003; 2005; Kim et al., 2007; Liston et al., 2007; Kim at al., 2009). Our recent studies of FoxpS expression and function provided critical novel insights into the biology of Treg cells and into cellular mechanisms of the immune homeostasis. Particularly relevant to the current grant application, was our finding that Treg cells utilize a subset of TCR distinct from that of effector T cells in the thymus and in the periphery (Hsieh et al., 2004; Hsieh et al., 2006). In agreement with studies of transgene-encoded TCR combined with transgene-encoded cognate ligand, we also found that TCR displayed by Treg cells exhibit a heightened reactivity towards self-peptide MHC class II complexes. The increased affinity TCR signals, together with additional signals, are essential for Treg cell differentiation and likely important for their function. The overall goal of this grant has been to explore a role for TCR signaling and specificity in Treg cell differentiation and function. Specifically, we have been addressing a role for MHC class II expressed on thymic epithelial cells and dendritic cells in generation of a functional repertoire of Treg cells (Aim 1 and 2), a role for dendritic and Treg cells, and TCR-MHC class II interactions in Treg cell maintenance in the periphery (Aim 2), and a role for TCR signaling in Treg cell differentiation and their ability to protect against immune- mediated inflammation (Aim 3).